1. Field of the Invention
The present invention concerns the identification on human eosinophils of the A3 adenosine receptor, and the use of compounds identified as specific modulators of adenosine's physiological actions to block activation of eosinophils. The pharmacology of compounds useful according to this invention is characterized through the use of cloned human adenosine A1, A2a, A2b and A3 receptor subtypes and functional assays. Compounds identified as antagonists of the A3 adenosine receptor subtype prevent the decrease in intracellular cAMP caused by activation of the A3 adenosine receptor by adenosine agonists. In this manner, A3 adenosine receptor specific antagonists are useful in preventing eosinophil activation and are therefore useful in the treatment or prevention of disease states induced by activation of the A3 adenosine receptor. These disease states include but are not limited to asthma, hypersensitivity, rhinitis, hay fever, serum sickness, allergic vasculitis, atopic dermatitis, dermatitis, psorasis, eczema, idiopathic pulmonary fibrosis, eosinophillic cholecystitis, chronic airway inflammation, hypereosinophilic syndromes, eosinophillic gastroenteritis, edema, urticaria, eosinophilic myocardial disease, episodic angioedema with eosinophilia, inflammatory bowel disease,ulcerative colitis, allergic granulomatosis, carcinomatosis, eosinophilic granuloma and familial histiocytosis.
2. Background
Adenosine is a naturally occurring nucleoside which exhibits diverse and potent physiological actions in the cardiovascular, nervous, pulmonary, renal and immune systems. Adenosine has been demonstrated to terminate superventricular tachycardia through blockage of atrioventricular nodal conduction (J. P. DiMarco, et al., (1985) J. Am. Col. Cardiol. 6:417-425, A. Munoz, et al., (1984) Eur. Heart J. 5:735-738). Adenosine is a potent vasodilator except in the kidney and placenta (R. A. Olsson, (1981) Ann. Rev. Physiol. 43:385-395). Adenosine produces bronchoconstriction in asthmatics but not in nonasthmatics (Cushly et al., 1984, Am. Rev. Respir. Dis. 129:380-384). Adenosine has been implicated as a preventative agent and in treatment of ventricular dysfunction following episodes of regional or global ischemia (M. B. Forman and C. E. Velasco (1991) Cardiovasc. Drugs and Therapy 5:901-908) and in cerebral ischemia(M. C. Evans, et al., (1987) Neurosci. Lett. 83:287, D. K. J. E., Von Lubitz, et al., (1988) Stroke 19:1133).
Dog A1 and A2a adenosine receptors were the first adenosine receptors to be cloned. See F. Libert, et al., (1989) Science 244:569-572, C. Maenhaut, et al., Biochem. Biophys. Res. Comm., (1990) 173:1169-1178, and F. Libert, et al. (1991) EMBO J. 10:1677-1682. The rat A1 adenosine receptor was cloned by L. C. Mahan, et al., (1991) Mol. Pharm. 40:1-7 and S. M. Reppert, et al., (1991) Mol. Endocrin. 5:1037-1048, the rat A2a by Fink et. al., (1992) Mol. Brain Res. 14:186-195, and the rat A2b by Stehle et al. (1992) Mol. Endocrinol. 6:384-393. Cloning of the rat A3 adenosine receptor was reported by Meyerhof et al., (1991) FEBS Lett. 284:155-160 and Zhou et al., (1992) PNAS U.S.A. 89:7432-7436. Cloning of the sheep A3 adenosine receptor has been reported by Linden et al., (1993) Mol. Pharm. 44:524-532. Cloning of the human A1, A2a, A2b and A3 receptors were reported in GB 2264948-A (Sep. 15, 1993). The human A1 adenosine receptor differs by 18 amino acids from the dog A1 sequence and 16 amino acids from the rat A1 sequence. The human A2a adenosine receptor differs by 28 and 71 amino acids, respectively from the dog and rat A2a sequences. The amino acid sequence for the human A3 receptor is 72% identical with the rat A3 receptor and 85% identical with the sheep A3 receptor sequences.
The actions of adenosine are mediated through the G-protein coupled receptors A1, A2a, A2b and A3 adenosine receptors. Upon activation of the adenosine receptors, the G-protein exerts either a stimulatory effect (A2a and A2b adenosine receptor coupled G-proteins) or an inhibitory effect (A1 and A3 adenosine receptor coupled G-proteins) on adenylate cyclase. Thus, adenosine receptor activation induces changes in intracellular cAMP and thereby initiates a cascade of intracellular events.
The adenosine receptors were initially classified into A1 and A2 subtypes on the basis of pharmacological criteria and coupling to adenylate cyclase (Van Caulker, D., Muller, M. and Hamprecht, B. (1979) J. Neurochem. 33, 999-1003.). Further pharmacological classification of adenosine receptors prompted subdivision of the A2 class into A2a and A2b subtypes on the basis of high and low affinity, respectively, for adenosine and the agonists NECA and CGS-21680 (Bruns, R. F., Lu, G. H. and Pugsley, T. A. (1986) Mol. Pharmacol. 29, 331-346; Wan, W., Sutherland, G. R. and Geiger, J. D. (1990) J. Neurochem. 55, 1763-1771). The existence of A1, A2a and A2b subtypes has been confirmed by cloning and functional characterization of expressed bovine, canine, rat and human receptors. A fourth subtype, A3, had remained pharmacologically undetected until its recent identification by molecular cloning. The rat A3 sequence, tgpcrl, was first cloned from rat testis by Meyerhoff et al. (see above). Subsequently, a cDNA encoding the identical receptor was cloned from striatum and functionally expressed by Zhou et al. (see above). When compared to the other members of the G-protein coupled receptor family, the rat sequence had the highest homology with the adenosine receptors (&gt;40% overall identity, 58% within the transmembrane regions). When stably expressed in CHO cells, the receptor was found to bind the radioligand .sup.125 I-APNEA (N.sup.6 -2-(4-amino-3-iodophenyl)ethyladenosine) and when transfected cells were treated with adenosine agonists, cyclic AMP accumulation was inhibited with a potency order of NECA=R-PIA&gt;CGS21680. The rat A3 receptor exhibited a unique pharmacology relative to the A1 and A2 adenosine receptor subtypes and was reported not to bind the xanthine antagonists 1,3-dipropyl-8-phenylxanthine (DPCPX) and xanthine amine congener (XAC). Messenger RNA for the rat A3 adenosine receptor is primarily expressed in the testis.
The sheep homolog of the A3 receptor was cloned from hypophysial pars tuberalis (see Linden et al. above). The sheep receptor is 72% identical to the rat receptor, binds the radioligand .sup.125 I-ABA and is also coupled to inhibition of cyclic AMP. The agonist affinity order of the sheep receptor is I-ABA&gt;APNEA&gt;NECA.gtoreq.R-PIA&gt;&gt;CPA. The pharmacology of xanthine antagonists was extensively studied and the sheep receptor was found to exhibit high affinity for 8-phenylxanthines with para-acidic substitutions. In contrast to the rat transcript, the expression of the sheep A3 adenosine receptor transcript is widespread throughout the brain and is most abundant in the lung and spleen. Moderate amounts of transcript are also observed in pineal and testis. Thus, because the published literature provides an inconsistent profile of adenosine A3 receptor pharmacology and tissue distribution, it has not been possible to predict the pharmacology or tissue distribution of the human A3 adenosine receptor.
The human A1, A2a and A2b adenosine receptor cDNAs have been cloned, and the tissue distribution of human adenosine receptor transcripts has been defined [Salvatore et al., P.N.A.S. 90:10365-10369, November 1993]. Based on the use of these cloned receptors, an assay has been described to identify adenosine receptor agonists, antagonists and enhancers and determine their binding affinity (see GB 2 264 948 A, published Sep. 15, 1993; see also R. F. Bruns, et al., (1983) Proc. Natl. Acad. Sci. U.S.A. 80:2077-2080; R. F. Brtms, et al., (1986) Mol. Pharmacol. 29:331-346; M. F. Jarvis, et al. (1989) J. Pharma. Exp. Therap. 251:888-893; K. A. Jacobson et al., (1989) J. Med. Chem. 32:1043-1051).
Adenosine receptor agonists, antagonists and binding enhancers have been identified and implicated for usage in the treatment of physiological complications resulting from cardiovascular, pulmonary, renal and neurological disorders. Adenosine receptor agonists have been identified for use as vasodilators ((1989) FASEB. J. 3(4) Abs 4770 and 4773, (19910 J. Med. Chem. (1988) 34:2570), antihypertensive agents (D. G. Taylor et al., FASEB J. (1988) 2:1799), and anti-psychotic agents (T. G. Heffner et al., (1989) Psychopharmacology 98:31-38). Adenosine receptor agonists have been identified for use in improving renal function (R. D. Murray and P. C. Churchill,(1985) J. Pharmacol. Exp. Therap. 232:189-193). Adenosine receptor allosteric or binding enhancers have shown utility in the treatment of ischemia, seizures or hypoxia of the brain (R. F. Bruns, et al. (1990) Mol. Pharmacol. 38:939-949; C. A. Janusz, et al., (1991) Brain Research 567:181-187). The cardioprotective agent, 5-amino-4-imidazole carboxamide (AICA) ribose has utility in the treatment of ischemic heart conditions, including unstable angina and acute myocardial infarction (H. E. Graber, et al. (1989) Circulation 80: 1400-1414).
8-phenylxanthines, methods of their synthesis and their use in human and veterinary therapy for conditions associated with the cell surface effects of adenosine have been described (EP 0 203 721, published Dec. 3, 1986). However, this publication is silent as to adenosine receptor subtypes and subtype specificity of disclosed compounds. In WO 90/00056, a group of 1,3-unsymmetrical straight chain alkyl-substituted 8-phenylxanthines were described as being potent bronchodilators. This disclosure is likewise silent as to the subtype specificity of disclosed compounds.
Through the use of homogenous, recombinant adenosine receptors, the identification and evaluation of compounds which have selectivity for a single receptor subtype has now been accomplished. Moreover, because of the variable effects of adenosine documented in other species, the utilization of human adenosine receptor subtypes is advantageous for the development of human therapeutic adenosine receptor agonists, antagonists or enhancers. The instant patent disclosure defines compounds which unexpectedly exhibit selective binding affinity for eosinophils by virtue of the presence on this cell type of the A3 adenosine receptor and therefore provides a method of using such compounds which overcomes the disadvantages of using compounds of uncharacterized specificity, by specifically blocking the activities mediated through the activation of the A3 receptor subtype without substantially blocking the activities of the other adenosine receptor subtypes.
Corticoidsteroids administered orally or through inhalation, are effective agents for the treatment of allergic, inflammatory and asthmatic states. Specifically, the use of corticoidsteroids for asthma reduces bronchial hyperresponsivness. The mechanism of action results from anti-inflammatory effects in part on eosinophils. The chronic use of corticoidsteroids results in a reduction in the number of eosinophils in the systemic circulation and within tissues, and in a decrease of the migration of eosinophils to sites of inflammation. A number of adverse systemic side effects are associated with the use of steroids including fluid retention, hypertension, peptic ulceration, and adrenal suppression. These side effects are the result of the multiple actions of steroids on tissues and organs. A eosinophil selective therapy would therefore, be advantageous for the reduction or elimination of these adverse effects.
Recently, the effectiveness of a monoclonal antibody raised against the integrin .alpha.4 chain was demonstrated in an allergic sheep model of airway late phase responses and airway hyperresponsivness (Abraham, et al. (1994) J. Clin. Invest. 93: 776-787). Administration of the blocking antibody either prior or after allergic challenge was found to be effective. The antibody was also found to reduce the platelet-activating factor induced peroxidase release from eosinophils and supported a mechanism of inhibiting eosinophil function for the alleviation of late phase responses and persisting airway hyperresponsiveness after antigen challenge. These data support the importance of eosinophil activation and degranulation in mediating allergic, asthmatic and hyperresponsive conditions and illustrates that agents which can modulate and inhibit eosinophil functions are effective in preventing and treating such conditions.
Salvatore et al., [P.N.A.S. 90:10365-10369, November 1993] disclosed the pharmacological profile of the human A3 adenosine receptor, and the tissue distribution of human A1, A2a, A2b and A3 adenosine receptor transcripts. In addition, the activity of certain substituted 8-phenyl xanthines as specific antagonists of the human A3 adenosine receptor was disclosed. The instant invention extends the characterization of the A3 adenosine receptor subtype by demonstrating the existence of the A3 adenosine receptor subtype on eosinophils. In view of the known association of intracellular cAMP decreases with eosinophil activation [see Kuehl, et al, (1987) Am. Rev. Respir. Dis. 136: 210-213; see also Kita et al., J. Immunol. 146:2712-2718, 1991, regulation of Ig-induced eosinophil degranulation by adenosine 3',5'-cyclic monophosphate], and the known coupling of the A3 adenosine receptor to inhibition of adenylate cyclase [Salvatore et al., P.N.A.S. 90:10365-10369, November 1993] and as confirmed herein, our discovery of the expression of the A3 adenosine receptor subtype in eosinophils demonstrates the utility of A3 adenosine receptor antagonists in the prevention of that component of eosinophil activation attributable to the decrease in intracellular cAMP normally induced by activation of the A3 adenosine receptor on eosinophils.